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ReadiUse™ Preactivated APC-AF700 Maleimide
APC-AF700 is a popular tandem color used in flow cytometry. Its primary absorption peak is at 651 nm with emission peak at ~700 nm. AAT Bioquest offers this preactivated APC-AF700 to facilitate the APC-AF700 tandem conjugations to reduced antibodies and other biomolecules that contain a thiol group. Our preactivated APC-AF700 maleimide is prepared from the commonly used crosslinker SMCC, and ready to conjugate. Allophycocyanin (APC) is a phycobiliprotein isolated from Spirulina sp., a blue-green alga. Like other phycobiliproteins, APC is fluorescent, with an extremely high absorptivity and a high quantum efficiency. It is a protein which can be easily linked to antibodies and other proteins by conventional protein cross-linking techniques without altering its spectral characteristics.
Example protocol
SAMPLE EXPERIMENTAL PROTOCOL
Reduction of Antibody
- Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µL) in distilled water. Antibody solutions should be at 2 mg/mL or higher for best results. The reduction can be carried out in different buffers for example: MES, phosphate, and TRIS buffers (pH range 6 to 8). The antibody should be concentrated if less than 2 mg/mL.
- Add 2 µL of 1.0 M DTT stock per 100 µL of antibody solution and mix well. Let the antibody solution stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
- Purify the reduced antibody over a desalting column pre-equilibrated with 50 mM MES Buffer (pH=6.0-6.5) with 2 mM EDTA. (Desalting column: https://www.aatbio.com/products/readiuse-bio-gel-p-6-spin-column?unit=60500)
- Measure the Antibody concentration with Nanodrop. (Con. (mg/ml)= A280nm/1.4) Note: The reduced antibody is not stable; the conjugation reaction needs to be carried out the conjugation as soon as possible after purification.
Conjugate with ReadiUse™ Preactivated APC-AF700 Maleimide
- Reconstitute ReadiUse™ Preactivated APC-AF700 Maleimide in 100 µL ddH2O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated APC-AF700 Maleimide are stable at 4 °C for one week, please kept it from light.
- Add reduced antibody to pre-activated APC-AF700 directly to at the ratio of 130 µg APC-AF700 /100 µg reduced antibody.
- Rotate the mixture for 60-120 mins at room temperature.
- After 60 minutes, block the free sulfhydryls on the antibody.
- Prepare a fresh solution of 10 mg/mL NEM in DMSO; add 3.4 µL per mg of antibody and rotate for 20 minutes at room temperature.
Purification
- Antibody/APC-AF700 conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/APC-AF700 conjugate solution is recommended to be stored at 2~8 °C and kept from light.
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
| ReadiUse™ Preactivated APC-Cy7 Maleimide | 651 | 779 | 700000 |
References
View all 8 references: Citation Explorer
Bivalent EGFR-Targeting DARPin-MMAE Conjugates.
Authors: Karsten, Lennard and Janson, Nils and Le Joncour, Vadim and Alam, Sarfaraz and Müller, Benjamin and Tanjore Ramanathan, Jayendrakishore and Laakkonen, Pirjo and Sewald, Norbert and Müller, Kristian M
Journal: International journal of molecular sciences (2022)
Authors: Karsten, Lennard and Janson, Nils and Le Joncour, Vadim and Alam, Sarfaraz and Müller, Benjamin and Tanjore Ramanathan, Jayendrakishore and Laakkonen, Pirjo and Sewald, Norbert and Müller, Kristian M
Journal: International journal of molecular sciences (2022)
Detecting and predicting neutralization of alemtuzumab responses in MS.
Authors: Saxena, Gauri and Moore, James M and Jones, Meleri and Pryce, Gareth and Ali, Liaqat and Leisegang, Georgia R and Vijay, Vivek and Loveless, Samantha and Robertson, Neil P and Schmierer, Klaus and Giovannoni, Gavin and Gnananpavan, Sharmilee and Baker, David and Tallantyre, Emma C and Kang, Angray S
Journal: Neurology(R) neuroimmunology & neuroinflammation (2020)
Authors: Saxena, Gauri and Moore, James M and Jones, Meleri and Pryce, Gareth and Ali, Liaqat and Leisegang, Georgia R and Vijay, Vivek and Loveless, Samantha and Robertson, Neil P and Schmierer, Klaus and Giovannoni, Gavin and Gnananpavan, Sharmilee and Baker, David and Tallantyre, Emma C and Kang, Angray S
Journal: Neurology(R) neuroimmunology & neuroinflammation (2020)
Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.
Authors: Bennett, Kristen and Sadler, Natalie C and Wright, Aaron T and Yeager, Chris and Hyman, Michael R
Journal: Applied and environmental microbiology (2016): 2270-2279
Authors: Bennett, Kristen and Sadler, Natalie C and Wright, Aaron T and Yeager, Chris and Hyman, Michael R
Journal: Applied and environmental microbiology (2016): 2270-2279
Pharmacokinetic Profile and Acute Toxicological Properties of a Novel Radiosensitizer Cytosine-Phosphate-Guanosine Oligodeoxynucleotide 107 in Mice Following Intravenous and Orthotopic Administration.
Authors: Cen, Yanyan and Li, Xiaoli and Yin, Zhiwei and Yan, Zifei and Liu, Dan and Peng, Wei and Pan, Feng and Zhou, Hong
Journal: Nucleic acid therapeutics (2015): 254-65
Authors: Cen, Yanyan and Li, Xiaoli and Yin, Zhiwei and Yan, Zifei and Liu, Dan and Peng, Wei and Pan, Feng and Zhou, Hong
Journal: Nucleic acid therapeutics (2015): 254-65
Membrane labeling of coral gastrodermal cells by biotinylation: the proteomic identification of surface proteins involving cnidaria-dinoflagellate endosymbiosis.
Authors: Li, Hsing-Hui and Huang, Zi-Yu and Ye, Shih-Png and Lu, Chi-Yu and Cheng, Pai-Chiao and Chen, Shu-Hwa and Chen, Chii-Shiarng
Journal: PloS one (2014): e85119
Authors: Li, Hsing-Hui and Huang, Zi-Yu and Ye, Shih-Png and Lu, Chi-Yu and Cheng, Pai-Chiao and Chen, Shu-Hwa and Chen, Chii-Shiarng
Journal: PloS one (2014): e85119
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